Candidate and Environment: This candidate has completed extensive neuroscience training at Harvard and MIT. Beth Israel Neurology has provided an independent laboratory and start-up funds. The candidate's K08 funding ends in 11/05 and he needs continued salary support to protect his research time. This Award would enable him to obtain the preliminary data necessary to obtain an R01 grant. His long term goal is to continue his full-time biomedical research career at Beth Israel/ Harvard Medical School. In the career development plan, the candidate proposes to apply his training and experience to the study of epilepsy. Research Project Summary: The molecular mechanisms in the brain that underlie the spike-and-wave seizures of childhood absence epilepsy (CAE) have long been debated. This proposal uses new bacteriophage P1-derived Cre/loxP recombination techniques to target absence epilepsy gene mutations to specific neuron subtypes in the murine brain. Childhood absence epilepsy-associated mutations were recently discovered in the T-type calcium channel Cav3.2 gene. The project's hypothesis is that Cav3.2 mutations alter the firing properties of specific neuron subtypes to cause the characteristic 3-5 Hz rhythmic discharge of spike-and-wave complexes, and the behavioral arrests afflicting children with absence epilepsy. To test this hypothesis, the candidate will recreate the disease in mice using an epitope-tagged CACNA1H transgene that encodes Cav3.2. Nucleotide mutations will be made to recreate the epilepsy-associated amino acid changes F161L and V831M, which alter Cav3.2 channel gating. Second, he will target the gene to specific neuron subtypes by adding a Cre recombinase delete-able trancriptional and translational silencing element to the transgene. Because they contain cell-type specific promoters, the Cre transgenes express Cre recombinase protein in limited neuron subtypes. Only in these neurons will Cre delete the silencing element, cause transgene expression, and generate epitope tag staining. In this study, the candidate will test whether abnormal burst firing in cortical pyramidal or reticular thalamic neurons cause absence epilepsy. If the characteristic signs of epilepsy are reproduced, it will establish that mutations in Cav3.2 cause absence epilepsy. Targeted expression of this mutant gene will identify the responsible neurons. Identifying the neural substrate will facilitate work to identify other disease genes and potential drug targets.